Il ruolo di Melusina nella funzione mitocondriale e nel metabolismo dei cardiomiociti derivanti da hiPSC

Melusin is a muscle-specific small co-chaperone protein that was originally discovered as an interactor of the integrin β1 cytoplasmic domain. Studies carried out in murine models demonstrated that Melusin is not required for the development and the physiological function of the heart, but plays a crucial role when the myocardium is exposed to stressful stimuli. Specifically, Melusin expression favors the activation of a beneficial compensatory hypertrophic response in murine hearts subjected to chronic pressure overload. The protein is key to cardiac mechanotransduction by interacting with integrins and co-operating with the scaffold protein IQGAP1 to form supramolecular complexes that include Hsp90, FAK, and the ERK1/2 MAPK signaling cascade. Recent evidence indicated that Melusin cardioprotective function also involves the regulation of cell metabolism. In murine hearts, Melusin inhibits the mitochondrial trifunctional protein, a key enzyme in fatty acid beta-oxidation. In physiological conditions the heart mainly relies on fatty acids to produce the huge amount of energy required for continuous beating. In absence of Melusin, lipid oxidation is further enhanced leading to the excessive reactive oxygen species (ROS) production. The present project aimed to develop an in vitro model of human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) for the study of Melusin protective function, and to investigate whether its role in cell metabolism is preserved in humans. First, we generated Melusin overexpressing (Mel-TG) and Knock-Out (Mel-KO) lines from wild type hiPSCs by molecular engineering; then we differentiated the edited lines into cardiomyocytes through a chemically-defined monolayer directed differentiation. Mel-KO hiPSCs differentiated at high efficiency into spontaneously beating cardiomyocytes, and the absence of Melusin mRNA and protein confirmed the successful knocking-out. We also obtained Mel-TG hiPSCs that constitutively expressed Melusin at high level, but for still unknown reasons these cell lines failed to differentiate into cardiomyocytes. Next, we assessed the mitochondrial functionality and metabolic activity of hiPSC-CMs. Mel-KO hiPSC-CMs showed an enhanced capability to oxidize palmitate, accompanied by increased mitochondrial function in terms of electron transport chain activity and ATP production. We also noticed a strict correlation between ROS levels and lipid metabolism. Interestingly, the ablation of Melusin in human cardiomyocytes had effects on sugar metabolism resulting in a drop of glucose utilization via TCA cycle. Overall, our data demonstrated a novel role for the co-chaperone protein Melusin in modulating fatty acid beta-oxidation, and demonstrated that this function is preserved in human cardiomyocytes.